The purpose of this project is to define hemostatic regulatory mechanisms of the brain. Because thrombo-occlusive processes are so important for stroke pathophysiology, brain regulation of hemostasis is a critical issue. Our work has been the first to show the hemostatic regulatory capacity of the blood-brain barrier, a function that we have termed "brain- specific hemostasis". We propose to further analyze this phenomenon by studying pericyte-endothelial and pericyte- astrocyte-endothelial interactions in blood-brain barrier models using human cells. These cells will be grown a) as monolayers; and b) in an artificial capillary network. We will continue to delineate the role of transforming growth factor-beta (TGF-beta) as a critical mediator of brain-specific hemostasis. We will also define the unique anticoagulant system within the brain that compensates for paucity of antithrombotic factors (eg, thrombomodulin) prevalent in the systemic vasculature. Candidates for endothelial antithrombotic molecules upregulated by astrocytes and pericytes include tissue factor pathway inhibitor, the protease nexins, prostacylin, and endothelial nitric oxide. Finally, we will analyze the role of shear stress on brain-specific hemostasis by subjecting a capillary network of endothelial cells, astrocytes, and pericytes to physiological and low shear stresses. Our findings will define pathways of brain regulation of hemostasis, add to the emerging field of vascular bed-specific hemostasis regulation, and pave the way for strategies to prevent and treat stroke by modulation of the brain's endogenous anticoagulant system.